Regulation of synthesis of phosphatidylcholine in mammalian cells can be controlled by CTP: phosphocholine cytidylyltransferase. This enzyme undergoes a reversible change in distribution from cytosolic to membrane-associated in cells made deficient in phosphatidylcholine. The proposed research is concerned with the structure and function of cytidylyltransferase in both its purified and cell-associated state. The quaternary structure of the purified enzyme will be determined by chemical cross-linking studies. The catalytic site and lipid-binding domains will be identified by affinity labeling techniques. The amino acid sequence of cytidylyltransferase will be determined by molecular cloning and sequencing a cDNA for the enzyme. The cDNA will also be used as a probe to examine tissue-specific expression of cytidylyltransferase as well as to screen genomic clones. These experiments will be performed with cytidylyltransferase isolated from rat liver as well as from rat brain. The subcellular location of cytidylyltransferase will be examined in control cultured cells and in cells that have been treated to deplete cellular phosphatidylcholine. Techniques to be used for the localization include immunofluorescence of intact cells and immunochemical analysis of membranes separated by gradient fractionation. Cytidylyltransferase localization will be examined in choline-starved cells, in which preliminary evidence indicates the enzyme is associated with the endoplasmic reticulum, as well as in phospholipase C-treated cells, where the enzyme does not appear to be in the endoplasmic reticulum. These experiments will thus examine in depth the structural aspects of cytidylyltransferase that allow it to be found associated with different cellular structures, as will identify those structures.